Bottom Line:
Consistent with its function in ES cells, cyclin H depletion from mouse embryos also leads to defects in the expansion of the inner cell mass of blastocysts, a transient pluripotent stem cell population in vivo.Our findings indicate that cyclin H has an essential function in promoting the self-renewal of the pluripotent stem cells of blastocyst stage embryos.Collectively, these studies demonstrate a critical and novel role for cyclin H in maintaining ES cell identity and suggest that cyclin H has important functions in early embryonic development.

Affiliation: Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACTThe trimeric Cdk7.cyclin H.Mat1 complex functions in cell cycle regulation, as the Cdk-activating kinase, and in transcription, as a module of the general transcription factor TFIIH. As a component of TFIIH, Cdk7 phosphorylates serines 5 and 7 of the carboxyl-terminal domain of RNA polymerase II and can also directly phosphorylate transcription factors to regulate gene expression. Here we have investigated the function of the Cdk7.cyclin H.Mat1 complex in murine embryonic stem (ES) cells and preimplantation embryos to determine whether it regulates the unique cell cycle structure and transcriptional network of pluripotent cells. We demonstrate that depletion of cyclin H leads to differentiation of ES cells independent of changes in cell cycle progression. In contrast, we observed that developmental genes are acutely up-regulated after cyclin H down-regulation, likely perturbing normal ES self-renewal pathways. We further demonstrate that Spt5, a known phosphorylation target of Cdk7, similarly regulates ES pluripotency and gene expression. Consistent with its function in ES cells, cyclin H depletion from mouse embryos also leads to defects in the expansion of the inner cell mass of blastocysts, a transient pluripotent stem cell population in vivo. Our findings indicate that cyclin H has an essential function in promoting the self-renewal of the pluripotent stem cells of blastocyst stage embryos. Collectively, these studies demonstrate a critical and novel role for cyclin H in maintaining ES cell identity and suggest that cyclin H has important functions in early embryonic development.

Mentions:
To analyze the cell cycle of ES cells upon cyclin H depletion, we performed flow cytometric analysis on cells labeled with BrdUrd and propidium iodide to determine the fraction of cells in the S phase. Surprisingly, cell cycle distribution was not altered upon cyclin H depletion, with the majority of cells in the S phase, as expected for ES cells (Fig. 5, A and B). When we analyzed cell cycle profiles at a later time point, there was a significant decrease in the fraction of cells in S phase with concomitant increases in both the G1 and G2 fractions (Fig. 5C; *, p < 0.05; **, p < 0.01). However, this change could be secondary to differentiation, because ES cells dramatically decrease proliferation upon the loss of pluripotency. Consistent with the cell cycle findings, we did not observe changes in the phosphorylation of Cdk1 (Thr161) or Cdk2 (Thr160) at the previously identified sites of Cdk activation by CAK (Fig. 5D). As an additional control, we immunoprecipitated Cdk2 and measured kinase activity toward histone H1. As expected, Cdk2 activity was not reduced after cyclin H depletion (Fig. 5E). Because cyclin H depletion did not completely abolish Cdk7 activity, it is possible that reduced levels of Cdk7 activity (Fig. 4F) are sufficient to maintain Cdk1 and Cdk2 phosphorylation (Fig. 5D). Several previously published reports argue for this interpretation. For example, depletion of Cdk7 expression itself by 75% does not reduce Cdk2 phosphorylation (50). Cdk2 may also be particularly insensitive to dephosphorylation in ES cells because it is constitutively associated with cyclin (49). Collectively, these findings suggest that significant depletion of cyclin H does not alter Cdk2 activity or cell cycle progression acutely in ES cells. Consequently, changes in cell cycle distribution observed after prolonged cyclin H depletion are likely to be secondary to the loss of pluripotency rather than a “driver” of differentiation.

Mentions:
To analyze the cell cycle of ES cells upon cyclin H depletion, we performed flow cytometric analysis on cells labeled with BrdUrd and propidium iodide to determine the fraction of cells in the S phase. Surprisingly, cell cycle distribution was not altered upon cyclin H depletion, with the majority of cells in the S phase, as expected for ES cells (Fig. 5, A and B). When we analyzed cell cycle profiles at a later time point, there was a significant decrease in the fraction of cells in S phase with concomitant increases in both the G1 and G2 fractions (Fig. 5C; *, p < 0.05; **, p < 0.01). However, this change could be secondary to differentiation, because ES cells dramatically decrease proliferation upon the loss of pluripotency. Consistent with the cell cycle findings, we did not observe changes in the phosphorylation of Cdk1 (Thr161) or Cdk2 (Thr160) at the previously identified sites of Cdk activation by CAK (Fig. 5D). As an additional control, we immunoprecipitated Cdk2 and measured kinase activity toward histone H1. As expected, Cdk2 activity was not reduced after cyclin H depletion (Fig. 5E). Because cyclin H depletion did not completely abolish Cdk7 activity, it is possible that reduced levels of Cdk7 activity (Fig. 4F) are sufficient to maintain Cdk1 and Cdk2 phosphorylation (Fig. 5D). Several previously published reports argue for this interpretation. For example, depletion of Cdk7 expression itself by 75% does not reduce Cdk2 phosphorylation (50). Cdk2 may also be particularly insensitive to dephosphorylation in ES cells because it is constitutively associated with cyclin (49). Collectively, these findings suggest that significant depletion of cyclin H does not alter Cdk2 activity or cell cycle progression acutely in ES cells. Consequently, changes in cell cycle distribution observed after prolonged cyclin H depletion are likely to be secondary to the loss of pluripotency rather than a “driver” of differentiation.

Bottom Line:
Consistent with its function in ES cells, cyclin H depletion from mouse embryos also leads to defects in the expansion of the inner cell mass of blastocysts, a transient pluripotent stem cell population in vivo.Our findings indicate that cyclin H has an essential function in promoting the self-renewal of the pluripotent stem cells of blastocyst stage embryos.Collectively, these studies demonstrate a critical and novel role for cyclin H in maintaining ES cell identity and suggest that cyclin H has important functions in early embryonic development.

Affiliation:
Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACTThe trimeric Cdk7.cyclin H.Mat1 complex functions in cell cycle regulation, as the Cdk-activating kinase, and in transcription, as a module of the general transcription factor TFIIH. As a component of TFIIH, Cdk7 phosphorylates serines 5 and 7 of the carboxyl-terminal domain of RNA polymerase II and can also directly phosphorylate transcription factors to regulate gene expression. Here we have investigated the function of the Cdk7.cyclin H.Mat1 complex in murine embryonic stem (ES) cells and preimplantation embryos to determine whether it regulates the unique cell cycle structure and transcriptional network of pluripotent cells. We demonstrate that depletion of cyclin H leads to differentiation of ES cells independent of changes in cell cycle progression. In contrast, we observed that developmental genes are acutely up-regulated after cyclin H down-regulation, likely perturbing normal ES self-renewal pathways. We further demonstrate that Spt5, a known phosphorylation target of Cdk7, similarly regulates ES pluripotency and gene expression. Consistent with its function in ES cells, cyclin H depletion from mouse embryos also leads to defects in the expansion of the inner cell mass of blastocysts, a transient pluripotent stem cell population in vivo. Our findings indicate that cyclin H has an essential function in promoting the self-renewal of the pluripotent stem cells of blastocyst stage embryos. Collectively, these studies demonstrate a critical and novel role for cyclin H in maintaining ES cell identity and suggest that cyclin H has important functions in early embryonic development.